Hydrogels represent physically or chemically crosslinked polymer structures which are able to absorb large amounts of water without their dissolution in the aqueous solution. Regarding the suitable rheological parameters, the hydrogels with their properties resemble living tissues. Hydrogels are used in form of scaffolds in replacements, or tissues regeneration in case of a tissue damage. The cell organization, cell proliferation or morphogenesis determination may be controlled through hydrogels. At the same time, the hydrogels represent a suitable energy source for the cells. These insoluble three dimensional nets enable the immobilization of biologically active agents (amino acids, peptides, drugs, enzymes, grow factors etc.) and their following controlled release in the desired concentration, time and space. Out of building components of hydrogels, biopolymers are preferred to synthetic polymers, especially where the final application aims at the area of tissue engineering or regenerative medicine and a high biocompatibility of the tested material has to be ensured (Slaughter V. B., Khurshid S. S., Fisher O. Z., Khademhosseini, Peppas, N. A. 2009. Adv Mater 21: 3307). Polysaccharides are suitable polymers thanks to their easy availability, a relatively low price, excellent biocompatibility, useful mechanical properties and a manifold structural or functional variability. The most often used polysaccharides for pharmaceutical and biomedical applications are:
Hyaluronic acid (HA) is a natural heteropolysaccharide of glycosaminoglycan kind, formed with D-glucuronic and N-acetyl-D-glucosamine subunit, mutually bound through β(1-3) and β(1-4) O-glycosidic bond. HA naturally appears in many connective tissues, synovial fluid, aqueous humour, skin and cartilages (Smeds K. A., Pfister-Serres A., Mild G., Dastqheib K., Inoue M., Hatchell D. L., Grinstaff M. W. 2001. J Biomed Mater Res 54: 115). Thanks to its biocompatibility, HA is utilized in biomedicine, nutrition, cosmetic and pharmaceutical industry.
Chondroitin sulfate (CS) is a glycosaminoglycan composed of sulfated N-acetylgalactosamine and D-glucuronic acid that is in the greatest amount present in the extracellular matrix of cartilage. CS participates in the articular metabolism and is used as a therapeutic means against degenerative arthritis. As food supplements (e. g. Hyalgel) it plays an important role in prevention of osteoarthrosis (Bottegoni C., Muzzarelli R. A. A., Giovannini F., Busilacchi A., Gigante A. 2014: CarbPol 109: 126).
Chitosan (CH) is a cationic homopolysaccharide prepared by deacetylation of chitin and is extracted from exoskeleton of sea crustaceans. As CH comes from a natural, renewable nontoxic and biodegradable source, it is regarded as an ecologically acceptable product. Its quality and properties are dependent on its purity and the degree of deacetylation (usually in the range 70-95%), further on the molecular weight and also on the crystallinity. CH is usually used as a hypocholesterolemic and bacteriostatic preparation, drug vehicle or material for cell scaffold formation (Pasqui D., De Cagna M., Barbucci, R. 2012. Polymers 4: 1517). Sodium carboxymethyl cellulose (CMCNa) is a hydrophilic cellulose derivative produced by alkylation of swollen cellulose (homopolymer of β-D-glucopyranose) with chloroacetic acid under basic conditions. CMCNa, in combination with various drugs or optionally co-excipients, in the form of medical devices (gauze, bandage, wound dressings) is used in the therapy of skin diseases. It is applied in treating of diabetic foot, skin ulcers, post-operative surgery wound, in toxic epidermal necrolysis and also as skin implants (Pasqui D., De Cagna M., Barbucci, R. 2012. Polymers 4: 1517).
Polysaccharides in their native form do not form hydrogels. For this reason, an additional modification of their physical properties is required. It is mainly the decrease of solubility and increase of stability in aqueous solution. One option is a chemical modification, through which the polarity of the polysaccharide chain is decreased e. g. by blocking of the carboxyl group resulting in ester formation (U.S. Pat. Nos. 4,851,521, 4,965,353) or by hydrophobization of the polar hydroxyl groups (WO1996/035720, WO2010/105582, U.S. Pat. No. 3,720,662).
The second option is a chemical crosslinking within the polysaccharide structure. The most used reactions leading to a chemical crosslinking include polymerization (Burdick J. A., Chung c., Jia X., Randolph M. A., Langer R. 2005. Biomacromolecules 6: 386), condensation reactions (WO2008014787, WO2009/108100, WO2011/069474), dimerization reactions (EP0554898B1, EP0763754A2, US006025444), cycloaddition reactions (CZ304072), optionally enzymatic reactions (CZ303879). Oxidative reactions of polysaccharides according to WO2011/069474 and WO2011/069475 may be used for the synthesis of polysaccharide precursors that are suitable for additional chemical modifications including crosslinking reactions. Dehydration reaction of these precursors was thus used for the preparation of α,β-unsaturated analogues (CZ304512). Deacetylation of polysaccharides according to U.S. Pat. No. 7,345,117 is used for the preparation of polyamino derivatives required e. g. for nucleophilic addition.
However, classical chemical crosslinking also has several important and indisputable drawbacks, i. e. uncontrollable propagation of chemical reaction, insufficient chemoselectivity, using of crosslinking agents and the necessity of an additional purification of the final products. The combination of the classical chemical polysaccharide crosslinking with the use of photoreactive linkers can successfully overcome the above limitations. The photoreactive linkers contain photoremovable protecting groups (PPG) built in their structure. The preparation of monofunctional photoremovable carbamate linkers can be carried out according to (Figueiredo R. M., Fröhlich R., Christmann M. 2006 J OrgChem 71: 4147) or (Werner T., Barrett A. G. M. 2006 J OrgChem 71:4302 or Furuta, T., Hirayma Y., Iwamura M. 2001. OrgLett 3: 1809) by a reaction of an excess of a bifunctional aminolinker with an acylation agent carrying PPG.
One example of the application of PPG is substrate masking from recognition in biological system in vitro or in vivo, the so-called triggering of the biological response to the presence of a specific agent. These masked substrates are called caged molecules and in case of the used PPG, the term caging groups (CG) is used. CG help mainly in biotechnology and cellular biology as their photocleavage takes place under mild conditions, rapidly, precisely and can be excellently controlled in time and space. The CG applications fall in the area of photolithographic creation of complex peptides, oligonucleotides or in the area of biologically active compounds release in cells or tissues (US2002/0016472).
Another practical example of PPG can be a chemical reaction of two involved functional groups that does not proceed as long as one of them is masked with a photoremovable protecting group (PPG). After the PPG removal, the original reactive group is restored and it reacts with the other participating group in the reaction mixture. The advantage of the two-step process, the installation and cleavage of PPG, thus enables the control of the chemical reaction course. If the substrate in the reaction mixture is masked (protected), the chemical reaction does not proceed. If the substrate is regenerated (released) in the reaction mixture, the chemical reaction proceeds. The amount or the concentration of the masked and released substrate can be determined by using the source of electromagnetic radiation, both in the time aspect (on-off switch, light impulse), and in the space aspect (focused light, laser, use of a pbotomask etc.). Another advantage of the photocleavage is that it can be reliably applied where other approaches of introduction of protecting groups fail. It applies for example for pH-sensitive or thermosensitive substrates, biomaterials and in in vitro or in vivo applications. The approach disclosed here thus enables to control the qualitative parameters (crosslink accuracy and density), as well as the quantity parameters (the whole volume vs part of the sample) of the crosslinked material. For this reason the final crosslinked product can reach from viscous solutions, through soft, to elastic gels.
The term photochemically controlled chemical reaction can represent not only a conjugation reaction or a reaction leading to immobilization or, in the contrary, to the release of the substrate from the carrier structure. This approach can also be applied to the formation of crosslinked polymer structures through the crosslinking reaction with the masked substrate, which is the subject matter of this patent document.
In the literature, there are more practical examples of PPGs that undergo photolysis (Green T. W. & Wuts P. G. M., 1999, John Wiley, 3rd edition). Photolysis (chemical cleavage) of chemical bonds in these groups is the result of the light quantum—photon absorption by the substrate molecule. Photochemical cleavage of the protecting group can be accomplished by a direct chromophore excitation after the absorption of a single photon with the desired energy or by a multiphoton absorption followed by an electron transfer to the protection group (US210/0207078). In case of ammines, the introduced protecting groups are carbamate functional groups. The most used PPGs are alkoxy, or alternatively nitro derivatives of aromatic alcohols (Klán P., Šolomek T., Bochet Ch. G., Blanc A, Givens R., Rubina M., Popik V., Kostikov A., Wirz J. 2013: ChemRev 113: 119; US2008/0009630, and also heteroaromatics of coumarin, quinoline, xanthan or thioxanthone type (US2002/0016472). The application of carbamate PPGs falls mainly in the area of combinatorial peptide synthesis or nucleic acids synthesis (Piggot A. M. & Karuzo P. 2005. Tetr Lett 46: 8241). Some more patent documents exist (US2013309706A1, US20008028630A1, US20060216324A1), that use photolysis for the surface modification of polymer materials, controlled release of a biologically active compound controlled or, in the contrary, its covalent immobilization to the polymer structure. However, the use of PPGs for the controlled polysaccharide crosslinking has not been published yet. Presumably, the reason is a combination of multiple factors including for example an insufficient molar absorption coefficient of the chosen PPG for the desired wavelength range, a low quantum yield of the photolysis, a slow substrate release, a low stability and hydrophobic character of the PPG, a formation of potentially toxic and absorbing disintegrative photolysis products, their consecutive competitive reaction with the released substrate or the biological material.